A Recombinant Ig Fragment (IgCw-γεκ) Comprising the Cγ1-Cε2–4 and Cκ Domains Is an Alternative Reagent to Human IgE

Abstract

Human IgE is useful for immunological assays, such as sensitization of FcεRI-positive cells and IgE measurement. In this study, we report the development of a recombinant Ig fragment, designated IgCw-γεκ, as an alternative reagent to human IgE. IgCw-γεκ (∼130 kDa) comprises two hybrid constant H chain regions (Cγ1-Cε2–4, each ∼53 kDa) and two constant κ L chains (Cκ, each ∼12 kDa) and lacks a V domain. The presence of Cγ1 instead of Cε1 within the H chain increased the production yield and facilitated assembly of the H and L chains. IgCw-γεκ was produced in cultured human embryonic kidney 293F cells, with a yield of ∼27 mg/l. IgCw-γεκ bound to human FcεRIαRs expressed on the surface of rat basophilic leukemia-2H3 cells. A β-hexosaminidase release assay revealed that the biological activity of IgCw-γεκ was comparable with that of IgE. The IgE concentration measured using IgCw-γεκ as a standard was similar to that measured using IgE as a standard. These results suggest that the IgCw-γεκ molecule retains the basic characteristics of IgE, but does not cross-react with Ags, making it an alternative to the IgE isotype references used in a variety of immunological assays.

The dispensability of VH-VL pairing and the indispensability of VL domain integrity in the IgG1 secretion process

“…In a previous study of an IgE-based fragment, we proposed the potential involvement of alternative assembly mechanisms, and chaperones other than BiP. Co-expressing C ε1-4 and C κ chains in HEK293F cells led to secretion of individual chains without covalent assembly ( Kim et al, 2022 ), whereas co-expressing the C γ1-3 and C κ chains resulted in assembly and secretion of IgCw-γκ ( Kim et al, 2019 ). Substituting the C ε1 domain in the Cε 1-4 chain with the C γ1 domain restored secretion of assembled IgE-like molecule, designated as IgCw-γεκ, with a size of ∼130 kDa ( Kim et al, 2022 ).…”

“…Co-expressing C ε1-4 and C κ chains in HEK293F cells led to secretion of individual chains without covalent assembly ( Kim et al, 2022 ), whereas co-expressing the C γ1-3 and C κ chains resulted in assembly and secretion of IgCw-γκ ( Kim et al, 2019 ). Substituting the C ε1 domain in the Cε 1-4 chain with the C γ1 domain restored secretion of assembled IgE-like molecule, designated as IgCw-γεκ, with a size of ∼130 kDa ( Kim et al, 2022 ). Exploring the assembly process of IgE, which remains largely uncharted, would be highly fascinating.…”

Standardizing In Vitro β-Lactam Antibiotic Allergy Testing with Synthetic IgE

“…The absence of reference materials for the measurement of β-lactam specific IgE poses a significant challenge. While purified human IgE used in quantitative and qualitative assays is monoclonal, it has inherent disadvantages such as undesired cross-reactivity and lack of selectivity . Conversely, establishing a serum bank from a diverse range of allergic patients with substantial levels of IgE antibodies is impractical due to limitations in size, reproducibility of serum pools, and the significant quantity of IgE-positive sera required for each specificity .…”

“…While purified human IgE used in quantitative and qualitative assays is monoclonal, it has inherent disadvantages such as undesired cross-reactivity and lack of selectivity. 15 Conversely, establishing a serum bank from a diverse range of allergic patients with substantial levels of IgE antibodies is impractical due to limitations in size, reproducibility of serum pools, and the significant quantity of IgE-positive sera required for each specificity. 16 Previous attempts to develop artificial human sera, such as chimeric adaptor molecules and specific bi-nanobodies, have involved animal immunization and lack the functional structure of IgE.…”